CN103739686B - Improve to plant products and the relevant albumen of quality-improving and encoding gene and application - Google Patents
Improve to plant products and the relevant albumen of quality-improving and encoding gene and application Download PDFInfo
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Abstract
The invention discloses a kind of albumen relevant to plant products raising and quality-improving and encoding gene and application.Albumen provided by the present invention is following (a) or (b): the protein that (a) aminoacid sequence shown in sequence in sequence table 1 forms；The aminoacid sequence of b protein that (a) is limited by () through the replacement of one or several amino acid residue and/or disappearance and/or interpolation, and to improve plant products and improve the protein that plant quality is relevant.It is demonstrated experimentally that albumen provided by the present invention can improve seed single plant yield, 100-grain weight, seed size, fat content and plant height.The present invention is significant in cultivating high yield and high oil plants new varieties.
Description
Technical field
The invention belongs to genetic engineering field, relate to a kind of albumen relevant to plant products raising and quality-improving and encoding gene and application.
Background technology
Along with the continuous growth of population, world food consumption figure, always above yield, be it is predicted that the year two thousand thirty grain demand will improve 30% to 40%, therefore only improved constantly the yield of cereal crops, could meet ever-increasing grain demand.Along with improving constantly of economic level, the requirement of grain quality is also being improved by people, it is necessary to edible quality is better, the food that nutrition is more rich.
Semen sojae atricolor is a kind of important cereal crops and oil crop, is also the source of the main edible protein of the mankind, the raw material of industry and health care medicine.Soybean seed contains the albumen of 40% and the oils and fats of 20%.In addition substantial amounts of biological active substances such as isoflavone, lecithin, vitamin E, saponins etc. it are also enriched in.In recent years, being consumed in of the Semen sojae atricolor rich in albumen, oil and other health-care components is continuously increased by people so that the supply increasing Semen sojae atricolor is extremely urgent.Although soybean yields is improving constantly, but comparing other chief crops, soybean yields also has very big growth potential, and the composition of oils and fats and albumen also has greatly improved space.Modern biotechnology the boundary between broken biological can realize reconfiguring of hereditary material, biological according to mankind's design improvement in advance, so as to meet the needs of the mankind.Biotechnology is combined with traditional breeding technology, it is possible to improve soybean yields and improvement soybean quality.Therefore, find the gene that can improve soybean yields and improvement soybean quality, for soybean heredity improvement and breed of variety, the supply increasing Semen sojae atricolor is had important practical significance.
Nuclear factor-Y(NF-Y) it is the transcription factor of a kind of downstream gene expression that interacts by regulatory factor.NF-Y transcription factor contains conservative narrow spectrum sequence, and the CCAAT box in this sequence and promoter in eukaryote region combines.In several species, on number, CCAAT box is all the Homologous gene sequences of high conservative.In all eukaryotes, CCAAT box transcriptional regulatory assembly is a cis-regulating element, and is present in the promoter region of the gene of about 30%.Gene expression pattern can also be tissue or phase specificity, it is also possible to is determined by other cis and trans acting factors, and is probably to the expression controlling gene ubiquitous by the promoter comprising CCAAT box.Multiple trans acting factors are associated with CCAAT box, but only nuclear factor NF-Y combines with these 5 nucleotide of CCAAT.In arabidopsis, NF-Y transcription factor LEC1 regulates and controls the expression of the target gene in seed storage protein gene and some other downstream, and overexpression LEC1 causes that the global upregulation of fatty acid synthesis gene is expressed.In Brassica campestris L, process LAN BnLEC1 and BnL1L gene can improve seed oil content 2%-20%.In Semen Maydis, process LAN ZmLEC1 gene can improve seed oil content 48%.NF-YB6 is class LEC1 gene, in arabidopsis can the sudden change of complementary LEC1, overlapping with LEC1 function.
Summary of the invention
It is an object of the invention to provide a kind of albumen relevant to plant products raising and quality-improving and encoding gene and application.
Protein provided by the present invention, name is called GmNFYB6L, derives from bean 29 in Semen sojae atricolor (Glycinemax (L.) Merr.) kind, is following (a) or (b):
A protein that () aminoacid sequence shown in sequence in sequence table 1 forms；
The aminoacid sequence of b protein that (a) is limited by () through the replacement of one or several amino acid residue and/or disappearance and/or interpolation, and to improve plant products and improve the protein that plant quality is relevant.
For the ease of the purification of GmNFYB6L albumen, the amino terminal of the protein that the amino acid residue sequence of sequence 1 forms or carboxyl terminal label as shown in the table can be connected in by sequence table.
Table: the sequence of label
| Label | Residue | Sequence |
| Poly-Arg | 5-6(is generally 5) | RRRRR |
| Poly-His | 2-10(is generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Protein in above-mentioned (b) can synthetic, it is possible to first synthesize its encoding gene, then carries out biological expression and obtain.The encoding gene of the protein in above-mentioned (b) can pass through the codon that will lack one or several amino acid residue in the DNA sequence shown in sequence in sequence table 2, and/or carries out the missense mutation of one or several base pair.
The nucleic acid molecules encoding described GmNFYB6L albumen falls within protection scope of the present invention.
Described nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；Described nucleic acid molecules can also be RNA, such as mRNA, hnRNA or tRNA etc..
In one embodiment of the invention, described nucleic acid molecules is specially the gene (called after GmNFYB6L) encoding described GmNFYB6L albumen；Described GmNFYB6L gene is following 1) to 4) in arbitrary described DNA molecular:
1) coded sequence is the DNA molecular shown in 41-721 position of sequence 2 in sequence table；
2) sequence is the DNA molecular shown in sequence 2 in sequence table；
3) under strict conditions with 1) or 2) DNA molecule hybridize that limits and the DNA molecular encoding described GmNFYB6L albumen；
4) with 1)-3) in the DNA molecular of arbitrary restriction there is more than 90% homology and encode the DNA molecular of described GmNFYB6L albumen.
Above-mentioned stringent condition can be with the solution of 6 × SSC, 0.5%SDS, hybridizes at 65 DEG C, and then with 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively washes film once.
Wherein, sequence 2 is made up of 851 nucleotide, and 41-721 position is ORF, the GmNFYB6L albumen shown in sequence 1 in polynucleotide.
Protection scope of the present invention is fallen within containing the recombinant vector of above-mentioned nucleic acid molecules, expression cassette, transgenic cell line or recombinant bacterium.
Described recombinant vector can be recombinant expression carrier, it is possible to for recombinant cloning vector.
Described recombinant expression carrier can use existing plant expression vector construction.Described plant expression vector includes double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment, such as pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derivative plant expression vector.Described plant expression vector also can comprise 3 ' end untranslated regions of exogenous gene, namely comprises polyadenylation signals and the DNA fragmentation of any other participation mRNA processing or gene expression.The bootable polyadenylic acid of described polyadenylation signals joins 3 ' ends of mRNA precursor.When using described gene constructed recombinant expression carrier, any enhancement mode, composing type, organizing specific type or inducible promoter can be added before its transcription initiation nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiquitin promoter (pUbi), stress induced promoter rd29A etc., they can be used alone or be combined use with other plant promoter；In addition, when using the gene constructed recombinant expression carrier of the present invention, it be also possible to use enhancer, including translational enhancer or transcriptional enhancer, these enhancer regions can be ATG initiation codon or neighboring region start codon etc., but must be identical with the reading frame of coded sequence, to ensure the correct translation of whole sequence.The source of described translation control signal and start codon is widely, it is possible to be natural, it is also possible to be synthesis.Translation initiation region can come from transcription initiation region or structural gene.For the ease of transgenic plant cells or plant being identified and screening, recombinant expression carrier used can be processed, enzyme or the gene of luminophor, the antibiotic marker thing with resistance or the anti-chemical reagent marker gene etc. of color change can be produced as added the coding can expressed in plant.Also can be not added with any selected marker, directly screen transformed plant with adverse circumstance.
In an embodiment of the present invention, the promoter starting described GmNFYB6L genetic transcription in described recombinant expression carrier is specially 35S promoter.
More specifically, described recombinant expression carrier is insert the recombiant plasmid that described GmNFYB6L gene obtains between attR1 and the attR2 site of pB2GW7 carrier.
Described expression cassette is by the promoter that can start described GmNFYB6L gene expression, described GmNFYB6L gene, and transcription terminator composition.
Described GmNFYB6L albumen, or described nucleic acid molecules, or the application that described recombinant expression carrier, expression cassette, transgenic cell line or recombinant bacterium are in arbitrary as follows falls within protection scope of the present invention:
(1) regulation and control plant products；
(2) regulation and control plant quality；
(3) regulation and control plant plant height；
(4) plant variety of selection-breeding output increased；
(5) plant variety that selection-breeding quality improves；
(6) plant variety that selection-breeding plant height increases.
In the application, described yield specifically may be embodied at least one in (b1)-(b3) as follows；Described quality specifically may be embodied in as follows (b4):
(b1) single plant yield of plant seed；
(b2) 100-grain weight of plant seed；
(b3) volume of plant seed；
(b4) fat content of plant seed.
In the present invention, described regulation and control plant products is embodied in: in described plant, if the expression of described GmNFYB6L albumen or its encoding gene is more high, then the single plant yield of described plant seed, 100-grain weight and volume are more big.Described regulation and control plant quality is embodied in: in described plant, if the expression of described GmNFYB6L albumen or its encoding gene is more high, then in the seed of described plant, the content of fat is more high.Described regulation and control plant plant height is embodied in: in described plant, if the expression of described GmNFYB6L albumen or its encoding gene is more high, then the plant height of described plant is more high.
In the present invention, the method of the plant variety that the method for the plant variety of described selection-breeding output increased, described selection-breeding quality improve, and the method for plant variety that described selection-breeding plant height increases, all specifically can include as parent, plant higher for described GmNFYB6L expressing quantity is carried out the step hybridized.
It is a further object to provide a kind of method cultivating transgenic plant.
The method of cultivation transgenic plant provided by the present invention, specifically can comprise the steps:
A) in purpose plant, import the encoding gene of described GmNFYB6L albumen, obtain expressing the transgenic plant of described encoding gene；
B) obtain from step a) gained transgenic plant compared with described purpose plant, there is following a1)-a3) transgenic plant at least one in purpose character:
A1) plant products improves；
A2) plant quality improves；
A3) plant plant height increases.
In the process, described yield is embodied at least one in (b1)-(b3) as follows；Described quality is embodied in (b4) as follows:
(b1) single plant yield of plant seed；
(b2) 100-grain weight of plant seed；
(b3) volume of plant seed；
(b4) fat content of plant seed.
Described GmNFYB6L albumen expression in described transgenic plant is higher than described purpose plant；Encoding the gene (i.e. GmNFYB6L gene) of described GmNFYB6L albumen be described gene is following 1) to 4) in arbitrary described DNA molecular:
1) coded sequence is the DNA molecular shown in 41-721 position of sequence 2 in sequence table；
2) sequence is the DNA molecular shown in sequence 2 in sequence table；
3) under strict conditions with 1) or 2) DNA molecule hybridize that limits and the DNA molecular encoding described GmNFYB6L albumen；
4) with 1)-3) in the DNA molecular of arbitrary restriction there is more than 90% homology and encode the DNA molecular of described GmNFYB6L albumen.
Above-mentioned stringent condition can be with the solution of 6 × SSC, 0.5%SDS, hybridizes at 65 DEG C, and then with 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively washes film once.
Described GmNFYB6L gene specifically can be imported in described purpose plant by any of the above-described described recombinant expression carrier, obtains described transgenic plant.Specifically can pass through to use the conventional biology methods such as Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, conductance, agriculture bacillus mediated, particle gun that described recombinant expression carrier converts plant cell or tissue, and the plant tissue of conversion is cultivated into plant.Agriculture bacillus mediated biological method such as grade is transformed in plant cell or tissue.
In above-mentioned application or method, described plant can be dicotyledon, it is possible to for monocotyledon.In the present invention, described plant is Semen sojae atricolor, is specially soybean varieties east agriculture 50.
Experiment proves, T2 recombinant expression carrier pB2GW7-GmNFYB6L soybean transformation containing DNA molecular shown in sequence 2 obtained is for the seed single plant yield of homozygous transgenic plant, 100-grain weight, seed size, fat content, and plant height is obviously higher than the Wild-type soy under the same terms.The present invention is significant in cultivating high yield and high oil plants new varieties.
Accompanying drawing explanation
Fig. 1 is the T2 PCR testing result for Semen sojae atricolor strain (TL1-TL5) plant of the GmNFYB6L transgenic that isozygotys.Wherein, M is molecular weight standard, stripe size from top to bottom is followed successively by 5000,3000,2000,1000,750,500bp, 250bp；Swimming lane 1-5 respectively TL1-TL5 strain；Swimming lane 6 is Semen sojae atricolor (Glycinemax (L.) Merr.) the kind east agriculture 50(WT of non-transgenic)；Swimming lane 7 is the blank doing template with water.Swimming lane 8 is recombinant vector pB2GW7-GmNFYB6L positive control.
Fig. 2 is the T2 PCR testing result for Semen sojae atricolor strain (CK1-CK5) plant turning pB2GW7 empty carrier of isozygotying.Wherein, M is molecular weight standard, clip size from top to bottom is followed successively by 2000,1000,750,500,250,100bp；Swimming lane 1-5 is CK1-CK5 strain plant respectively；Swimming lane 6 is Semen sojae atricolor (Glycinemax (L.) Merr.) the kind east agriculture 50(WT of non-transgenic)；Swimming lane 7 is the blank doing template with water.Swimming lane 8 is recombinant vector pB2GW7-GmNFYB6L positive control.
Fig. 3 is the relative expression quantity measurement result of genes of interest GmNFYB6L in each soybean heredity material.Wherein, A is that T2 is for the relative expression quantity measurement result (with the expression of reference gene β-actin for 1) of genes of interest GmNFYB6L in the blade of GmNFYB6L genetically engineered soybean strain (TL1-TL5) that isozygotys.WT represents Semen sojae atricolor (Glycinemax (L.) Merr.) the kind east agriculture 50 of non-transgenic.B is the relative expression quantity measurement result (with the expression of reference gene β-actin for 1) of endogenous GmNFYB6L in the seed of Semen sojae atricolor (Glycinemax (L.) Merr.) the kind east agriculture 50 of non-transgenic and blade.
Fig. 4 is the T2 phenotypic evaluation result for GmNFYB6L genetically engineered soybean strain of isozygotying.Wherein, CK represents that T2 turns the Semen sojae atricolor strain CK1 of pB2GW7 empty carrier for isozygotying；GmNYFB6L represents that T2 is for isozygotying GmNFYB6L genetically engineered soybean strain TL3.
Detailed description of the invention
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all commercially obtain.
In following embodiment, T0 represents and shows the plant converting the present age；T1 represents and shows the seed that T0 produces for selfing and the plant grown up to by it；T2 represents and shows the seed that T1 produces for selfing and the plant grown up to by it.
Semen sojae atricolor (Glycinemax (L.) Merr.) kind east agriculture 50: be recorded in " PazMM; MartinezJC; KalvigAB; FongerTM, WangK.Improvedcotyledonarynodemethodusinganalternativeex plantderivedfrommatureseedforefficientAgrobacterium-medi atedsoybeantransformation.PlantCellRep.2006;25:206 213 " literary composition, the public can obtain from Inst. of Oil Crops, Chinese Academy of Agriculture.
PB2GW7 carrier: containing lethal gene ccdB in the T-DNA fragment of carrier pB2GW7, its both sides are containing the LR recognition sequence reacted.This carrier is purchased from VIB(network address: http://www.psb.ugent.be/gateway).
Agrobacterium tumefaciems GV3101: being recorded in " AmandaMDavis; AnthonyHall; AndrewJMillar; ChiarinaDarrahandSethJDavis; Protocol:Streamlinedsub-protocolsforfloral-diptransforma tionandselectionoftransformantsinArabidopsisthaliana; 2009,5:310.1186/1746-4811-5-3 " literary composition, the public can obtain from Inst. of Oil Crops, Chinese Academy of Agriculture.
Gateway test kit: for invitrogen Products, its catalog number is 11789-100.Test kit has BP reaction enzymes mixture (BPClonaseTMAnd LR reaction enzymes mixture (LRClonase enzymemixture)TMEnzymemixture).
PDONR-Amp carrier: as carrier (donorvector).It is recorded in " MingLi; BoDing; JunbinWang; WenliYang; RuiWang; ShuguangBao, SilinZhong, XiaodongXie.WheatTaWRKY10-1isinvolvedinbiologicalrespons estothesalinityandosmostressesintransgenicArabidopsispla nts.AustralianJournalofCropScience.AJCS7 (6): 723-729 (2013) " " thepDONR (Amp') vector " literary composition in a literary composition, the public can obtain from Inst. of Oil Crops, Chinese Academy of Agriculture.
The acquisition of embodiment 1, soybean protein GmNFYB6L and encoding gene thereof
Take the tender seed of the children of bean 29 Post flowering 15d in Semen sojae atricolor (Glycinemax (L.) Merr.) kind of field growing and extract total serum IgE, and reverse transcription obtains cDNA, with this cDNA for template, under the guiding of primer PF and primer PR, expand by Standard PCR method.
PF:5 '-CGAGGCTCTTATAATCACACACAC-3 ' (the 1-24 position of sequence 2)；
PR:5 '-ACTCCCTTCCCAGCCCTT-3 ' (reverse complementary sequence of the 834-851 position of sequence 2).
Pcr amplification product is carried out 1% agarose gel electrophoresis detection after terminating by reaction.Reclaim also purification and be about the DNA fragmentation of 850bp, carry out sequencing.
Sequencing result shows, the sequence of gained PCR primer is sequence 2 in sequence table, is GmNFYB6L by this unnamed gene.The 41-721 position of sequence 2 is its open reading frame, the protein (called after GmNFYB6L) shown in sequence 1 in polynucleotide.
Embodiment 2, the acquisition of GmNFYB6L genetically engineered soybean and qualification
One, the acquisition of recombinant expression carrier pB2GW7-GmNFYB6L and qualification
The GmNFYB6L gene (sequence 2) that embodiment 1 is obtained by LR reaction is adopted to be connected to pB2GW7 carrier, it is thus achieved that recombinant vector pB2GW7-GmNFYB6L.Concrete operations are as follows:
1, the acquisition of attB-PCR product
(1) with the GmNFYB6L gene (sequence 2) of embodiment 1 acquisition for template, carrying out pcr amplification with primer attB-GmNFYB6L-F and primer attB-GmNFYB6L-R, its product (being designated as attB-1-PCR product) is purified after terminating by reaction.
(2) with step (1) gained attB-1-PCR product for template, carrying out pcr amplification with primer attB-adapter-F and primer attB-adapter-R, its product (being designated as attB-PCR product) is purified after terminating by reaction.
AttB-GmNFYB6L-F:5 '-GCAGGCTTCCGAGGCTCTTATAATCACACACAC-3 ' (underscore part is intermediate carrier pDONR-Amp sequence, and sequence thereafter is the 1-24 of sequence 2)；
AttB-GmNFYB6L-R:5 '-AGCTGGGTCACTCCCTTCCCAGCCCTT-3 ' (underscore part is intermediate carrier pDONR-Amp sequence, and sequence thereafter is the reverse complementary sequence of the 834-851 position of sequence 2)；
AttB-adapter-F:5 '-GGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3 ' (underscore part is intermediate carrier pDONR-Amp sequence, and sequence thereafter is consistent with underscore part in attB-GmNFYB6L-F primer)；
AttB-adapter-R:5 '-GGGGACCACTTTGTACAAGAAAGCTGGGTC-3 ' (underscore part is intermediate carrier pDONR-Amp sequence, and sequence thereafter is consistent with underscore part in attB-GmNFYB6L-R primer)；
2, BP reaction
BP reaction system:
25 DEG C of temperature bath reaction 16h.
BP reacts screening and the qualification of genes of interest clone, as follows:
1) taking BP product 5 μ l and convert the competent cell of bacillus coli DH 5 alpha, 37 DEG C are inverted cultivation 12-16h(culture medium is Amp resistance)；
2) picking monoclonal, 37 DEG C of 180-200rpm shake bacterium and cultivate (additional 50 μ g/mlAmp)；
3) extract plasmid, with M13 primer (M13F:5 '-TGTAAAACGACGGCCAGT-3 '；M13R:5 '-CAGGAAACAGCTATGACC-3 ') or gene specific primer (PF:5 '-CGAGGCTCTTATAATCACACACAC-3 '；PR:5 '-ACTCCCTTCCCAGCCCTT-3 ') inspection positive colony.
4) by the clone after order-checking is compared with original series (sequence 2), to show between attP1 and attP2 of pDONR-Amp carrier the recombiant plasmid called after pDONR-Amp-GmNFYB6L of DNA fragmentation shown in sequence 2 in insertion sequence table through order-checking, recombiant plasmid pDONR-Amp-GmNFYB6L is the entry vector of GmNFYB6L gene.
3, LR reaction
The entry vector pDONR-Amp-GmNFYB6L of Semen sojae atricolor GmNFYB6L gene step 2 obtained carries out LR reaction, and reaction system is as follows:
25 DEG C of temperature bath 16h.
LR reacts screening and the qualification of genes of interest clone, as follows:
1) taking LR product 5 μ l and convert the competent cell of escherichia coli DH5a, 37 DEG C are inverted cultivation 12-16h(culture medium is Spectinomycin resistance)；
2) picking monoclonal, 37 DEG C of 180-200rpm shake bacterium and cultivate (additional 50 μ g/ml spectinomycins)；
3) extract plasmid, with gene specific primer (PF:5 '-CGAGGCTCTTATAATCACACACAC-3 '；PR:5 '-ACTCCCTTCCCAGCCCTT-3 ') detection positive colony.
4) by the clone after order-checking is compared with original series (sequence 2), will show to insert the recombiant plasmid called after pB2GW7-GmNFYB6L of DNA fragmentation shown in sequence 2 between attR1 and the attR2 site of pB2GW7 carrier through order-checking.In recombinant expression carrier pB2GW7-GmNFYB6L, the promoter starting described GmNFYB6L genetic transcription is 35S promoter.
Two, the acquisition of GmNFYB6L genetically engineered soybean and qualification
1, the acquisition of restructuring Agrobacterium tumefaciems
Recombinant expression carrier pB2GW7-GmNFYB6L step one obtained converts Agrobacterium tumefaciems GV3101 by freeze-thaw method, to the recombinational agrobacterium after converting with by primer PF and PR(sequence ibid) primer pair that forms carries out PCR qualification.By the identified Agrobacterium GV3101 called after GV3101/pB2GW7-GmNFYB6L shown containing GmNFYB6L gene (stripe size of PCR order is about 851bp).
The comparison proceeding to pB2GW7 empty carrier is set simultaneously.The Agrobacterium GV3101 called after GV3101/pB2GW7 of pB2GW7 empty carrier will be proceeded to.
2, the acquisition of GmNFYB6L genetically engineered soybean
Utilize two kinds of recombinational agrobacterium GV3101/pB2GW7-GmNFYB6L and GV3101/pB2GW7 that step 1 obtains, respectively with agriculture bacillus mediated cotyledon node regeneration method soybean transformation (Glycinemax (L.) Merr.) kind east agriculture 50, obtaining the T2 Semen sojae atricolor strain 5 for homozygous transgenic GmGmNFYB6L, T2 is 5 for the Semen sojae atricolor turning empty carrier of isozygotying.Concrete grammar is as follows:
(1) recombinational agrobacterium GV3101/pB2GW7-GmNFYB6L or GV3101/pB2GW7 is taken, containing rifampicin 30 μ g/ml, the flat lining out of YEP solid medium of gentamycin (50 μ g/ml) and spectinomycin (100 μ g/ml), picking monoclonal be inoculated in 2ml contain in corresponding antibiotic YEP fluid medium 28 DEG C cultivate 24-28 hour after add fresh YEP fluid medium in the ratio (volume ratio) of 1:1000, 28 DEG C of shaking tables are cultured to OD600 and are about 1.2-1.5, it is centrifuged and removes supernatant postprecipitation equal-volume fluid medium (formula: 1/10B5 salt+B5 organic+MES5.9g/L+ sucrose 30g/L+ acetosyringone 30mg/L+6-BA1.67mg/L+GA30.25mg/L, pH5.4.The concentration of each material is respective substance final concentration in the medium) resuspended, in 28 DEG C, 140rpm cultivate 30 minutes stand-by.
(2) seed coat will be removed after the seed disinfection of Semen sojae atricolor (Glycinemax (L.) Merr.) kind east agriculture 50, remove hypocotyl and be about 3mm, rip cutting cotyledonary node, remove the terminal bud of connexon leaf segment, obtain outer implant.Outer implant being inserted suspends in the Agrobacterium of step (1) contaminates 30 minutes, proceed to after draining Agrobacterium in solidified co-cultivation medium, plane upwards, 24 DEG C, 18 h light/6 h dark are cultivated 3-5 days, proceed in inducing culture, plane upwards, every ware 8-10 outer implant, 25 DEG C, 18 h light/6 h dark are cultivated 14 days, proceed in fresh inducing culture, cultivate 14 days.Removing cotyledon, hypocotyl removes one layer, reserves fresh wounds, is placed in elongation medium, within every 2 weeks, changes a subculture, condition of culture isogeneous induction.The branch base portion of elongation 2-3 centimetre is immersed aseptic IBA(1mg/ml) the solution several seconds, it is transferred in root media.Shift in flowerpot after taking root.Grow after 2-3 sheet Newborn Leaves until seedling, be applied in leaf table, screening resistance Seedling (T0 generation) with the careless fourth phosphine solution that concentration is 100mg/ml.
Wherein, the formula of solidified co-cultivation medium: 1/10B5 salt (a great number of elements+trace element)+B5 vitamin+2-morpholino ethyl sulfonic acid (MES) 5.9g/L+ sucrose 30g/L+ acetosyringone 30mg/L+ bis-sulfur sulfydryl threitol (DTT) 150mg/L+L-cysteine 150mg/L+6-benayl aminopurine (6-BA) 1.67mg/L+ gibberellins (GA3) 0.25mg/L+ fungistat (bacterium presses down) 100mg/L, agar 4.5g/L, pH5.4.The concentration of each material is respective substance final concentration in the medium.
The formula of inducing culture: B5 medium 3.2g/L+ agar 8g/L+ sucrose 30g/L+MES0.59g/L, pH5.8, after sterilizing, add cephamycin 200mg/L, Ticarcillin/Clavulanate Acid (tim) 100mg/L, vancomycin (van) 50mg/L, bacterium presses down 200mg/L, grass fourth phosphine (PPT) 5mg/L, 6-benzyl aminopurine (BA) 1.1mg/L.
The formula of elongation medium: MS salt (a great number of elements+trace element)+B5 vitamin+agar 8g/L+ sucrose 30g/L+MES0.59g/L, pH5.8, after sterilizing, adds cephamycin 200mg/L, Ticarcillin/Clavulanate Acid 100mg/L, vancomycin 50mg/L, aspartic acid 50mg/L, L-Glutimic acid 50mg/L, bacterium presses down 200mg/L, grass fourth phosphine 5mg/L, anti-ribosylzeatin 0.5mg/L, gibberellins 0.5mg/L.
The formula of root media: MS salt (a great number of elements+trace element)+B5 vitamin+agar 8g/L+ sucrose 20g/L+MES0.59g/L, pH5.7.
3, the qualification of GmNFYB6L genetically engineered soybean
(1) PCR identifies
The qualification of A.GmNFYB6L genetically engineered soybean
The blade of resistance Seedling (T0 generation) the GmNFYB6L genetically engineered soybean obtained from step 2 extracts DNA, with primer Bar-F and Bar-R, Bar gene is carried out pcr amplification, purpose product is sized to 450bp, amplified production is carried out 1% agarose gel electrophoresis, the plant obtaining 450bp band is designated as the positive.
Bar-F:5 '-GAAGTCCAGCTGCCAGAAAC-3 '；
Bar-R:5 '-AAGCACGGTCAACTTCCGTA-3 '.
To be accredited as positive T0 for planting seed in greenhouse through above PCR, extract DNA in blade and carry out PCR qualification (method is ibid) after growing first trifoliolate leaf, after the positive strain of screening, results T1 is for seed.Plantation screening T1 gathers in the crops T1 for tied T2 on individual plant each in strain for seed for seed individual plant after the same method.Choose self progeny and all there is the T2 of glyphosate resistance for plant, be T2 for the GmNFYB6L genetically engineered soybean strain isozygotied.
Obtain 5 T2 altogether for isozygotying GmNFYB6L genetically engineered soybean strain, be designated as TL1-TL5 respectively.
Further, GmNFYB6L genetically engineered soybean strain (TL1-TL5) that 5 T2 generations of gained isozygotied adopts as above method to carry out PCR Molecular Identification.Three above comparison is set simultaneously: compare using Semen sojae atricolor (Glycinemax (L.) Merr.) the kind east agriculture 50 of non-transgenic as wild type (WT)；Using water as blank；Using plasmid pB2GW7-GmNFYB6L as positive control.
Result shows: TL1-TL5 strain plant all amplifies the purpose band that size is about 450bp, consistent with positive control；And the plant of the Semen sojae atricolor of the non-transgenic as wild type control (Glycinemax (L.) Merr.) kind east agriculture 50 amplifies corresponding purpose band for being with blank, as shown in Figure 1.
B. the qualification of pB2GW7 empty carrier Semen sojae atricolor is turned
The resistance Seedling (T0 generation) obtained from step 2 turns the blade of pB2GW7 empty carrier Semen sojae atricolor and extracts DNA, with primer 35S-F and 35S-R, 35S promoter is carried out pcr amplification, purpose product is sized to 250bp, amplified production is carried out 1% agarose gel electrophoresis, the plant obtaining 250bp band is designated as the positive.
35S-F:5 '-ACTAGAGCCAAGCTGATCTC-3 '；
35S-R:5 '-TGTCGTGCTCCACCATGTTG-3 '.
To be accredited as positive T0 for planting seed in greenhouse through above PCR, extract DNA in blade and carry out PCR qualification (method is ibid) after growing first trifoliolate leaf, after the positive strain of screening, results T1 is for seed.Plantation screening T1 gathers in the crops T1 for tied T2 on individual plant each in strain for seed for seed individual plant after the same method.Choose self progeny and all there is the T2 of glyphosate resistance for plant, be T2 for what isozygoty and turn pB2GW7 empty carrier Semen sojae atricolor strain.Obtain 5 T2 altogether and turn pB2GW7 empty carrier Semen sojae atricolor strain for isozygotying, be designated as CK1-CK5 respectively.
Further, for isozygotying, 5 T2 of gained being turned pB2GW7 empty carrier Semen sojae atricolor strain (CK1-CK5) adopts as above method to carry out PCR Molecular Identification.Three above comparison is set simultaneously: compare using Semen sojae atricolor (Glycinemax (L.) Merr.) the kind east agriculture 50 of non-transgenic as wild type (WT)；Using water as blank；Using plasmid pB2GW7-GmNFYB6L as positive control.
Result shows: CK1-CK5 strain plant all amplifies the purpose band that size is about 250bp, consistent with positive control；And the plant of the Semen sojae atricolor of the non-transgenic as wild type control (Glycinemax (L.) Merr.) kind east agriculture 50 amplifies corresponding purpose band for being with blank, as shown in Figure 2.
(2) real-time fluorescence quantitative PCR detection
Taking the T2 generation that step (1) obtains isozygotys GmNFYB6L genetically engineered soybean strain (TL1-TL5), T2 is for Semen sojae atricolor (Glycinemax (L.) Merr.) the kind east agriculture 50(WT of isozygoty the Semen sojae atricolor strain (CK1-CK5) turning pB2GW7 empty carrier and non-transgenic) plant, total serum IgE is extracted respectively from blade, reverse transcription obtains cDNA, with this cDNA for template, with special primer F1 and R1, the cDNA of gene GmNFYB6L is carried out real-time fluorescence quantitative PCR amplification, with Semen sojae atricolor β-actin for internal reference, primer is FC and RC.
Real-time fluorescence quantitative PCR reaction system: 2 × SuperRealPreMix (Tian Gen company) 10 μ L, forward primer (10uM) 0.6 μ L, reverse primer (10uM) 0.6 μ L, cDNA2 μ L, RNase-freeddH2O to 20 μ L.
Real-time fluorescence quantitative PCR response procedures: 95 DEG C of 15min；95 DEG C of 10sec, 60 DEG C of 20sec, 72 DEG C of 20sec, 40 circulations；Melt curve analysis analysis rises to 90 DEG C from 50 DEG C, and each step rises 1 DEG C, and the predissolve condition of the first step waits 90sec, and later each step waits 5sec.
Real-time fluorescence quantitative PCR is at StepOnePlusTMCarrying out on real-time fluorescence quantitative PCR instrument, a parallel test sets 3 repetitions.Utilize LivakKJ and SchmittgenTD(LivakKJSchmittgenTD.AnalysisofRelativeGeneE xpressionDataUsingReal-TimeQuantitativePCRandthe22DDCTMe thod.METHODS, 2001:25,402 408) method reported, i.e. 2-ΔΔCTCalculate relative expression quantity.Δ Δ CT=(CTGmNFYB6L-CTβ-actin) Timex-(CTGmNFYB6L-CTβ-actin) Time0；Timex represents random time point, and Time0 represents that the target gene of 1 times amount is expressed after β-actin corrects.
The sequence of above-mentioned primer is as follows:
F1:5 '-GCTTAACTCTCAGTAATTGGTGCT-3 ' (the 770-793 position of sequence 2)；
R1:5 '-ACTCCCTTCCCAGCCCTTT-3 ' (reverse complementary sequence of the 833-851 position of sequence 2)；
FC:5 '-ATTGGACTCTGGTGATGGTG-3 '；
RC:5 '-TCAGCAGAGGTGGTGAACAT-3 '.
Test in triplicate, results averaged.
In result such as Fig. 3 shown in A, Semen sojae atricolor (Glycinemax (L.) Merr.) the kind east agriculture 50(WT of non-transgenic) plant blade in substantially can't detect the expression of genes of interest GmNFYB6L (in the Wild-type soy of non-transgenic, GmNFYB6L is only specific expressed in the seed grown, blade is not expressed substantially, in GmNFYB6L transgenic plant, because 35S promoter be constitutive promoter, so the expression of GmNFYB6L can be detected in blade.As detailed below)；And the T2 that step (1) obtains is significantly high for the expression of genes of interest GmNFYB6L in the blade of the Semen sojae atricolor strain TL1-TL5 of the GmNFYB6L transgenic that isozygotys.T2 is essentially identical with WT for the result of the Semen sojae atricolor strain (CK1-CK5) turning pB2GW7 empty carrier of isozygotying, no difference of science of statistics.
Semen sojae atricolor (Glycinemax (L.) Merr.) kind east agriculture 50(WT) seed and blade in GmNFYB6L gene real-time fluorescence quantitative PCR detection, specific as follows:
Take Semen sojae atricolor (Glycinemax (L.) Merr.) kind east agriculture 50(WT), from seed and blade, extract total serum IgE respectively, reverse transcription obtains cDNA, with this cDNA for template, carries out real-time fluorescence quantitative PCR detection according to method ibid.In result such as Fig. 3 shown in B, it can be seen that at Semen sojae atricolor (Glycinemax (L.) Merr.) kind east agriculture 50(WT) in, endogenous GmNFYB6L specific gene expression is in seed, and does not substantially express in blade.
Embodiment 3, GmNFYB6L genetically engineered soybean phenotypic evaluation
In the T2 generation obtained with embodiment 2, isozygotys the Semen sojae atricolor strain (TL1-TL5) of GmNFYB6L transgenic, and T2 generation isozygotys Semen sojae atricolor (Glycinemax (L.) Merr.) the kind east agriculture 50(WT of the Semen sojae atricolor strain (CK1-Ck5) turning pB2GW7 empty carrier and non-transgenic) plant is experiment material.The seed (seed of every kind of material takes 20) of each material is seeded in greenhouse, the phenotype of each plant is observed when identical management and cultivation, gather in the crops seed after maturation and survey the single plant yield of each plant seed, 100-grain weight, fat content and protein content (concrete operations are referring to instrument operation instructions) in seed is measured with corn near-infrared analyzer (FOX company of Denmark, INSTRUMENT MODEL 1241).Experiment in triplicate, takes three times the meansigma methods repeated for quantitative data result.
Result shows: compared with the Semen sojae atricolor strain (CK1-CK5) turning pB2GW7 empty carrier of isozygotying with T2 generation, T2 increases for the plant height of the Semen sojae atricolor strain (TL1-TL5) of the GmNFYB6L transgenic that isozygotys, seed volume increases (Fig. 4).Additionally, with the Semen sojae atricolor of non-transgenic (Glycinemax (L.) Merr.) kind east agriculture 50(WT) compared with, T2 significantly improves (table 1) for the seed single plant yield of Semen sojae atricolor strain (TL1-TL5) of GmNFYB6L transgenic that isozygotys, 100-grain weight, fat content.Each parameter of observed statistical analysis above, T2 generation isozygoty turn pB2GW7 empty carrier Semen sojae atricolor strain (CK1-CK5) all with the Semen sojae atricolor of non-transgenic (Glycinemax (L.) Merr.) kind east agriculture 50(WT) basically identical, no difference of science of statistics.
The measurement result of each strain hundred grain weight of table 1, single plant yield, fat content and protein content
| Strain is numbered | 100-grain weight (gram) | Single plant yield (gram) | Fat content (%) | Protein content (%) 10--> |
| WT | 9.4±1.3 | 3.1±0.8 | 17.3±0.6 | 44.2±0.7 |
| TL1 | 16.4±1.5** | 3.44±0.3 | 19.7±0.5* | 42.8±0.5* |
| TL2 | 17.3±1.1** | 5.37±0.6** | 23.8±0.8** | 37.7±0.3** |
| TL3 | 14.6±1.0** | 5.55±0.8** | 22.7±0.4** | 35.9±0.3** |
| TL4 | 18.7±1.3** | 7.31±0.4** | 22.07±0.3** | 41.9±0.7* |
| TL5 | 15.9±1.2** | 4.14±0.3* | 23.6±0.5** | 39.4±0.6** |
Note: * represents compared with WT, and P < 0.05 is notable；* represents compared with WT, and P < 0.001 is notable.
Claims (14)
1. protein, the protein that the aminoacid sequence shown in sequence in sequence table 1 forms.
2. the nucleic acid molecules of protein described in coding claim 1.
3. nucleic acid molecules according to claim 2, it is characterised in that: described nucleic acid molecules is the gene of protein described in coding claim 1；Described gene is following 1) or 2) described in DNA molecular:
1) coded sequence is the DNA molecular shown in 41-721 position of sequence 2 in sequence table；
2) sequence is the DNA molecular shown in sequence 2 in sequence table.
4. contain the recombinant vector of nucleic acid molecules described in Claims 2 or 3.
5. contain the expression cassette of nucleic acid molecules described in Claims 2 or 3.
6. contain the recombinant bacterium of nucleic acid molecules described in Claims 2 or 3.
7. recombinant vector according to claim 4, it is characterised in that: described recombinant vector is recombinant expression carrier or recombinant cloning vector.
8. recombinant vector according to claim 7, it is characterised in that: in described recombinant expression carrier, the promoter transcribed starting described gene is 35S promoter.
9. the protein described in claim 1 or the nucleic acid molecules described in Claims 2 or 3 or the recombinant vector described in claim 4 or 7 or 8 or the expression cassette described in claim 5 or the recombinant bacterium described in claim 6 application in arbitrary as follows:
(1) regulation and control plant plant height；
(2) regulation and control plant leaf blade shape；
(3) plant variety that selection-breeding plant height increases；
(4) plant variety that selection-breeding blade shape changes.
10. application according to claim 9, it is characterised in that: described plant is dicotyledon.
11. application according to claim 10, it is characterised in that: described dicotyledon is Semen sojae atricolor.
12. cultivate and there is following a1) and the method for a2) at least one in purpose character transgenic plant, comprise the steps:
A) in purpose plant, import the encoding gene of protein described in claim 1, obtain expressing the transgenic plant of described encoding gene；
B) from step a) gained transgenic plant, obtain, compared with described purpose plant, there is following a1) and a2) at least one in purpose character transgenic plant:
A1) plant plant height increases；
A2) plant leaf blade alteration of form.
13. method according to claim 12, it is characterised in that: described plant is dicotyledon.
14. method according to claim 13, it is characterised in that: described dicotyledon is Semen sojae atricolor.
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